Bacillus thuringiensis ("B.t.") is a gram-positive soil bacterium that produces proteinaceous crystalline inclusions during sporulation. These B.t. crystal proteins are often highly toxic to specific insects. Insecticidal activities have been identified for crystal proteins from various B.t. strains against insect larvae from the insect orders Lepidoptera (caterpillars), Diptera (mosquitos, flies) and Coleoptera (beetles).
Recently certain B.t. strains and B.t. crystal proteins have been reported as having activity against non-insect species such as nematodes. The term "insecticidal," as used herein with reference to B.t. strains and their crystal proteins, is intended to include such pathogenic activities against non-insect species.
Individual B.t. crystal proteins, also called delta-endotoxins or parasporal crystals or toxin proteins, can differ extensively in their structure and insecticidal activity. These insecticidal proteins are encoded by genes typically located on large plasmids, greater than 30 megadaltons (mDa) in size, that are found in B.t. strains. A number of these B.t. toxin genes have been cloned and the insecticidal crystal protein products characterized for their specific insecticidal properties. A good review of cloned B.t. toxin genes and crystal proteins is given by Hofte et al., Microbiol. Rev. 53:242-255 (1989) (hereinafter Hofte and Whiteley, 1989), who also propose a useful nomenclature and classification scheme that has been adopted in this disclosure.
The insecticidal properties of B.t. have been long recognized, and B.t. strains were first commercially introduced in biological insecticide products in the 1960's. Commercialized B.t. insecticide formulations typically contain dried B.t. fermentation cultures whose crystal protein is toxic to various insect species and, in the past, were derived from "wild-type" B.t. strains, i.e., purified cultures of B.t. strains isolated from natural sources.
Several newly commercialized B.t. strains are genetically altered strains that have increased insecticidal potency as well as insecticidal activity against a broader spectrum of target insects, as compared with the parent B.t. strains. Such strains are exemplified in International Patent Publication No. WO 88/08877, published Nov. 17, 1988 by applicant Ecogen Inc. and in its counterpart U.S. Pat. No. 5,080,897 issued to Gonzalez, Jr. et al. on Jan. 14, 1992.
Development of these genetically altered B.t. strains did not involve recombinant DNA technology but was instead based on the techniques of plasmid conjugal transfer, which is a natural form of genetic exchange between bacteria, and of plasmid curing, in which certain nonessential plasmids are deleted from a bacterium.
Plasmid conjugal transfer, or conjugation, is limited by the fact that many plasmids carrying useful toxin genes are not amenable to transfer from their native host B.t. strain to another "recipient" B.t. strain. Furthermore, some plasmids which can be transferred by conjugation are inherently incompatible with other plasmids, so a stable "transconjugant" B.t. strain, containing the two desired, incompatible plasmids, cannot be constructed.
Another drawback to conjugation is that some mobilizable, or transferable, plasmids carry undesirable toxin genes in addition to the desired gene, so the quantity of the desired crystal protein produced is limited by concurrent production of an unwanted crystal protein.
Despite the demonstrated efficacy of commercialized transconjugant B.t. strains against certain target insects, there is a clear need for improved B.t. strains against other insect pests. Development of such B.t. strains will be facilitated by use of recombinant DNA technology in B.t. strain construction.
Recombinant DNA procedures provide great flexibility in the construction of novel plasmids containing one or more toxin genes, by permitting selection, manipulation and control of crystal protein type and production and of gene regulation and expression. Some techniques for utilizing the recombinant DNA approach in the production of transformed B.t. strains are described in European Patent Application Publication No. EP 0 342 633, published Nov. 23, 1989 by applicant Ciba-Geigy AG, and in European Patent Application Publication No. 0 537 105, published Apr. 14, 1993 by applicant Sandoz Ltd.
The recombinant B.t. strains disclosed in EP 0 342 633, EP 0 537 105 and other publications are generally characterized by the presence of one or more antibiotic resistance marker genes on the recombinant plasmid harboring the desired B.t. toxin gene(s). Such antibiotic resistance marker genes provide a means for the identification and selection of transformed B.t. strains containing the recombinant toxin-encoding plasmid but are undesirable in viable B.t. strains developed for use in commercial insecticide formulations. Since antibiotic resistance genes are not ordinarily present in native B.t. strains, pesticide and environmental regulatory agencies may be reluctant to approve antibiotic-resistant recombinant B.t. strains for unrestricted environmental release and for use in biological insecticide formulations.
A major reason for the presence of antibiotic resistance genes in recombinant B.t. strains described in the literature is the use of bifunctional cloning vectors containing such resistance marker genes. Portions of these cloning vectors are typically derived from plasmids not native to B.t., e.g., Escherichia coli, Bacillus cereus, Bacillus subtilis or Staphylococcus aureus plasmids, and contain, in addition to the antibiotic resistance marker gene, an origin of replication from a non-B.t. source that is also functional in B.t. and therefore permits the cloning vector to be replicated and maintained in B.t.
International Patent Publication No. WO 91/18102, published Nov. 28, 1990 by applicant Ecogen Inc., describes a plasmid shuttle vector for recombinant B.t. strain development that facilitates incorporation of recombinant plasmids into B.t. strain constructs that contain no DNA derived from E. coli or other non-B.t. biological sources. Using this shuttle vector, a cloned B.t. toxin gene and B.t. plasmid replication origin region are isolated as a single restriction fragment that, upon self-ligation, is introduced into B.t. by cotransformation, This plasmid shuttle vector utilizes a B.t. replication origin derived from large resident plasmids of B.t., a multiple cloning site and strategically placed restriction endonuclease cleavage sites to enable construction of B.t. strains that are free of antibiotic resistance marker genes and free of non-B.t. replication origins.
A second approach for constructing such B.t. strains is a multistep technique described by Lereclus et al., Bio/Technology 10:418-421 (1992) that relies on the presence of IS232 in a resident B.t. toxin plasmid to effect homologous recombination. A cloned B.t. toxin gene is inserted within a cloned fragment of IS232 (which is found on some naturally-occurring toxin-encoding B.t. plasmids) that is inserted into a shuttle plasmid thermosensitive for replication in B.t. The shuttle plasmid is then used to transform a B.t. strain containing the IS232 fragment on a resident B.t. plasmid, and transformants are selected at non-permissive temperature for clones in which the shuttle vector has integrated via homologous recombination into a copy of IS232 present on the resident plasmid. Subsequently, individual clones are screened for a second homologous recombination event that eliminates the shuttle vector and conserves the newly introduced toxin gene. This technique is limited by the laborious nature of its steps and its reliance on homologous recombination using IS232-containing resident B.t. plasmids, whose copy number cannot readily be altered to increase gene expression.
Removal of unwanted selectable marker genes or other unwanted DNA has been described for transgenic plants and eukaryotic cells via the so-called Cre/lox recombination system of bacteriophage P1, where the cre gene encoding the Cre recombinase enzyme is activated to delete the unwanted DNA, which is bracketed by Iox recombination site sequences. International Patent Publication No. WO 93/01283, published Jan. 21, 1993 by applicant U.S. Department of Agriculture, and Dale et al., "Gene transfer with subsequent removal of the selection gene from the host genome," Proc. Natl. Acad. Sci. USA 88:10558-10562 (1991), describe such a system for removal of a antibiotic resistance marker gene from transgenic tobacco plants.
U.S. Pat. No. 4,959,317 issued Sep. 25, 1990 to Sauer describes the application of the Cre/lox recombination system to yeast cells and to a mouse cell line to delete or invert selected DNA sequences.
Hofte and Whiteley, 1989, in discussing factors such as conjugative plasmid transfer that account for the observed mobility of crystal protein genes among B.t. strains, note past reports of some cryIA-type genes and the cryIVB gene being associated with insertion sequence (IS) elements on transposon-like structures (see paragraph bridging pages 245-246). Nevertheless, the role of repeat sequence and/or insertion sequence elements and transposon-like structures in the mobility of B.t. crystal protein genes still remains speculative.
Among known B.t. strains, only one transposon (transposable element) has been reported in the literature as having been isolated from B.t. Mahillon et al., EMBO J. 7:1515-1526 (1988) provide a detailed description of this transposon, originally reported in a 1983 publication and now named Tn4430. Murphy, "Transposable Elements in Gram-Positive Bacteria," Chapt. 9 in Mobile DNA, Berg et al., eds., Am. Soc. Microbiol., Washington, D.C. (1989) pp. 269-288, likewise discusses Tn4430, in the context of other transposable elements found in gram-positive bacteria.
Mahillon et al., Plasmid 19:169-173 (1988), describe the cloning in E. coli and restriction mapping of three small cryptic plasmids from B.t. var. thuringiensis, one of the plasmids being pGI2 which was reported to contain the B.t. transposon Tn4430. The authors speculate (at page 173) that the cloned plasmids could serve as the starting point for the development of new shuttle vectors for E. coli and B.t. but offer no details concerning the construction and use of such hypothetical plasmid shuttle vectors. The complete nucleotide sequence of the small cryptic plasmid pGI2, including Tn4430, is reported by Mahillon et al. in Nucl. Acids Res. 16:11827-11828 (1988).
Earlier references cited by Mahillon et al. in EMBO J. 2:1515-1526 (1988) disclose that, although Tn4430 is widely distributed among B.t. species, the functional role of Tn4430 in B.t., if any, remains unclear. Despite occasional mention in investigative research publications concerning B.t., of Tn4430 and of homology of its elements with other known insertion sequence elements, this transposon has not been utilized to facilitate construction of insecticidal B.t. strains; see, e.g., Lereclus et al., FEMS Microbioi. Lett. 49:417-422 (1988).
The novel transposon of the present invention, designated Tn5401, is only the second transposon to be isolated from B.t. since the discovery of Tn4430 over ten years ago. Unlike Tn4430 which is widely distributed among B.t. species, transposon Tn5401 appears to be found in only a few relatively rare B.t. species.
The present invention also encompasses a site-specific recombination system for recombinant B.t. strain construction that preferably utilizes certain elements of transposon Tn5401, e.g., its internal resolution site and recombinase gene. The site-specific recombination system of this invention represents a significant advance over the approach described in International Patent Publication No. WO 91/18102 because it facilitates the rapid development and construction of recombinant B.t. strains whose recombinant plasmids possess highly desirable characteristics. They are completely free of foreign DNA from non-B.t. sources and can carry B.t. toxin genes that provide insecticidal properties superior to B.t. strains presently used in commercial bioinsecticides.